The present invention is in the field of animal health, and is directed to vaccine compositions and diagnostics for disease. More particularly, the present invention relates to polynucleotide molecules comprising nucleotide sequences encoding GRA1, GRA2, SAG1, MIC1, and MAG1 proteins from Neospora, which polynucleotide molecules and proteins are useful in the production of vaccines against neosporosis, and as diagnostic reagents.
Neospora is a pathogenic protozoan parasite of animals that has been recognized as a major cause of abortion, neonatal death, congenital infection, and encephalitic disease in mammals. Dubey and Lindsay, 1996, Vet. Parasitol. 67:1-59; Dubey and Lindsay, 1993, Parasitology Today, 9:452-458. Neospora caninum infects dogs, and congenitally infects pups, often leading to paralysis. Tachyzoites of N. caninum have been isolated from naturally infected pups. Lindsay and Dubey, 1989, J. Parasitol. 75:163-165. Neospora is a major cause of abortion in dairy and beef cattle. Cases of Neospora-related disease, i.e., neosporosis, have also been reported in goats, sheep and horses.
Although N. caninum is superficially similar to the pathogen, Toxoplasma gondii, N. caninum and T. gondii have been distinguished from each other both antigenically and ultrastructurally. Dubey and Lindsay, 1993, above. In addition, Neospora-like protozoan parasites isolated from the brains of aborted bovine fetuses and continuously cultured in vitro were shown to be antigenically and ultrastructurally distinct from both T. gondii and Hammondia hammondi, and were most similar to N. caninum. Conrad et al., 1993, Parasitology 106:239-249. Furthermore, analysis of nuclear small subunit ribosomal RNA genes revealed no nucleotide differences between strains of Neospora isolated from cattle and dogs, but showed consistent differences between Neospora and T. gondii. Marsh etal., 1995, J. Parasitol. 81:530-535.
The etiologic role of a bovine isolate of Neospora in bovine abortion and congenital disease has been confirmed. Barr et aL, 1994, J. Vet. Diag. Invest. 6:207-215. A rodent model of central nervous system neosporosis has been developed using inbred BALB/c mice infected with N. caninum. Lindsay et al., 1995, J. Parasitol. 81:313-315. In addition, models to study transplacental transmission of N. caninum in pregnant outbred and inbred mice have been described by Cole et aL, 1995, J. Parasitol. 81:730-732, and by Long et al., 1996, J. Parasitol. 82:608-611, respectively. An experimental N. caninum pygmy goat model that closely resembles naturally acquired Neospora-induced cattle abortion has been demonstrated. Lindsay et al., 1995, Am. J. Vet. Res. 56:1176-1180. An experimental N. caninum sheep model that closely resembles naturally acquired Neospora-induced cattle abortion has also been demonstrated. Buxton et al., 1997, J. Comp. Path. 117:1-16.
In T. gondii, electron dense granules comprising an excretory-secretory group of antigens are present in the cytoplasm of tachyzoites. These antigens have been designated as GRA proteins. The GRAL protein of T. gondii has been reported to have a molecular weight ranging from about 22-27 kDa, and the GRA2 protein of T. gondii has been reported to have a molecular weight of about 28 kDa. Sam-Yellowe, 1996, Parasitol. Today 12:308-315. Similar electron dense granules are present in the cytoplasm of N. caninum tachyzoites (Bjerkas et al., 1994, Clin. Diag. Lab. Immunol. 1:214-221; Hemphill et aL, 1998, Intl. J. Parasitol. 28:429-438).
T. gondii cells are also known to comprise a group of major surface antigens that have been designated as SAG. The SAG1 protein of T. gondii is reported to have a molecular weight of about 30 kDa. Kasper et al., 1983, J. Immunol. 130:2407-2412. Monoclonal antibodies directed against T. gondii SAG1 protein significantly blocked the ability of T. gondii tachyzoites to invade bovine kidney cells under tissue culture conditions. Grimwood and Smith, 1996, Intl. J. Parasitol. 26: 169-173. Because T. gondii SAG1 appears to play a role in the invasion process, it has been hypothesized that SAG1 may be necessary to support the virulence phenotype. Windeck and Gross, 1996, Parasitol. Res. 82:715-719. Consistent with this hypothesis is the observation that mice immunized with T. gondii SAG1 protein and then challenged with T. gondii had reduced toxoplasma cyst formation in their brains than did control mice. Debard et al., 1996, Infect. Immun., 64:2158-2166. T. gondii SAG1 may be functionally related to a similar molecule in N. caninum designated as NC-p36 described by Hemphill et al., 1997, Parasitol. 115:371-380.
Micronemes are intracelluar organelles located at the apical end of tachyzoites of both T. gondii and Neospora, and may play a role in host cell recognition and attachment to the host cell surface during invasion. Formaux etal., 1996, Curr. Top. Microbiol. Immunol. 219:55-58. At least 4 different microneme-associated (MIC) proteins have been identified in T. gondii. The MIC1 protein of T. gondii is about 60 kDa, binds to the surface of host cells, and has been reported to have partial homology to thrombospondin-related adhesive protein (TRAP) from Plasmodium falciparum which binds to human hepatocytes. Robson et al. 1995 EMBO J. 14:3883-3894.
The conversion of parasites from tachyzoites to bradyzoites is critical for chronic infection and persistence of T. gondii. A gene expressing an immunodominant, bradyzoite-specific 65 kD antigen, designated as MAG1, has been identified in T. gondii. Parmley et a/., 1994, Mol. Biochem. Parasitol. 66:283-296. MAG1 has been reported to be specifically expressed in bradyzoite cysts, but not in the tachyzoite stage. This specificity of expression may indicate the involvement of MAG1 in the conversion between tachyzoite and bradyzoite stages of the life cycle of the parasite. Bohne et al., 1996, Curr. Topics Microbiol. Immunol. 219:81-91.
Identification in Neospora of protein homologs of T. gondii GRA1, GRA2, SAG1, MIC1, and MAG1 proteins, and the nucleotide sequence of polynucleotide molecules encoding said Neospora proteins, will serve to facilitate the development of vaccines against neosporosis, as well as diagnostic reagents.
The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the GRA1 protein from N. caninum. In a preferred embodiment, the GRA1 protein has the amino acid sequence of SEQ ID NO:2. In a further preferred embodiment, the isolated GRA1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO:1 from about nt 205 to about nt 777, the nucleotide sequence of the open reading frame (ORF) of the GRA1 gene, which is presented in SEQ ID NO:3 from about nt 605 to about nt 1304, and the nucleotide sequence of the GRA1-encoding ORF of plasmid pRC77 (ATCC 209685). In a non-limiting embodiment, the isolated GRA1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:3. The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a GRA1-encoding polynucleotide molecule of the present invention. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the GRA1 protein of N. caninum. The present invention further provides a polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned GRA1-related polynucleotide molecules.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the GRA2 protein from N. caninum. In a preferred embodiment, the GRA2 protein has the amino acid sequence of SEQ ID NO:5. In a further preferred embodiment, the isolated GRA2-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of the ORF of SEQ ID NO:4, which is from about nt 25 to about nt 660, and the nucleotide sequence of the GRA2-encoding ORF of plasmid pRC5 (ATCC 209686). In a non-limiting embodiment, the isolated GRA2-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:4. The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a GRA2-encoding polynucleotide molecule of the present invention. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the GRA2 protein of N. caninum. The present invention further provides a polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned GRA2-related polynucleotide molecules.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the SAG1 protein from N. caninum. In a preferred embodiment, the SAG1 protein has the amino acid sequence of SEQ ID NO:7. In a further preferred embodiment, the isolated SAG1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of the ORF of SEQ ID NO:6, which is from about nt 130 to about nt 1089, and the nucleotide sequence of the SAG1-encoding ORF of plasmid pRC102 (ATCC 209687). In a non-limiting embodiment, the isolated SAG1-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:6. The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a SAG1-encoding polynucleotide molecule of the present invention. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the SAG1 protein of N. caninum. The present invention further provides a polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned SAG 1-related polynucleotide molecules.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the MIC1 protein from N. caninum. In a preferred embodiment, the MIC1 protein has the amino acid sequence of SEQ ID NO:9. In a further preferred embodiment, the isolated MIC1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of the ORF of SEQ ID NO:8 from about nt 138 to about nt 1520, the nucleotide sequence of the ORF of the MIC1 gene, which is presented as SEQ ID NO:10, and the nucleotide sequence of the MIC1-encoding ORF of plasmid pRC340 (ATCC 209688). In a non-limiting embodiment, the isolated MIC1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO:8, and the nucleotide sequence of SEQ ID NO:10. The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a MIC1-encoding polynucleotide molecule of the present invention. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the MIC1 protein of N. caninum. The present invention further provides a polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned MIC1-related polynucleotide molecules.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the MAG1 protein from N. caninum. The MAG1 protein has a putative amino acid sequence shown in SEQ ID NO:13. In a preferred embodiment, the isolated MAG1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence presented in SEQ ID NO:11 from about nt 1305 to about nt 2786, a cDNA molecule prepared therefrom, such as a cDNA molecule having the ORF of SEQ ID NO:12 from about nt 122 to about nt 1381, and the nucleotide sequence of the MAG1-encoding ORF present in plasmid bd304 (ATCC 203413). The present invention further provides a polynucleotide molecule having a nucleotide sequence of any ORF present in SEQ ID NO:11. In a non-limiting embodiment, the isolated MAG1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:12. The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a MAG1-encoding polynucleotide molecule of the present invention. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the MAG1 protein of N. caninum. The present invention further provides a polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned MAG1-related polynucleotide molecules.
The present invention further provides a polynucleotide molecule comprising the nucleotide sequence of the promoters of the N. caninum GRA1 and MAG1 genes, which is presented in SEQ ID NO:11 from about nt 127 to about nt 703, and includes its complementary sequence.
The present invention further provides oligonucleotide molecules that hybridize to any of the polynucleotide molecules of the present invention, or that hybridize to a polynucleotide molecule having a nucleotide sequence that is the complement of any of the polynucleotide molecules of the present invention.
The present invention further provides compositions and methods for cloning and expressing any of the polynucleotide molecules of the present invention, including recombinant cloning vectors, recombinant expression vectors, transformed host cells comprising any of said vectors, and novel strains or cell lines derived therefrom. More particularly, the present invention provides a recombinant vector comprising a polynucleotide molecule having a nucleotide sequence encoding the GRA1, GRA2, SAG1, MIC1 or MAG1 protein of N. caninum. In specific, though non-limiting, embodiments, the present invention provides plasmid pRC77 (ATCC 209685) encoding GRA1; plasmid pRC5 (ATCC 209686) encoding GRA2; plasmid pRC102 (ATCC 209687) encoding SAG1; plasmid pRC340 (ATCC 209688) encoding MIC1; and plasmid bd304 (ATCC 203413) comprising the MAG1 gene sequence and the MAG1IGRA1 bidirectional promoter region.
The present invention further provides a substantially purified or isolated N. caninum polypeptide selected from the group consisting of GRA1, GRA2, SAG1, MIC1 and MAG1 proteins. In a preferred embodiment, the N. caninum GRA1 protein has the amino acid sequence of SEQ ID NO:2. In another preferred embodiment, the N. caninum GRA2 protein has the amino acid sequence of SEQ ID NO:5. In another preferred embodiment, the N. caninum SAG1 protein has the amino acid sequence of SEQ ID NO:7. In another preferred embodiment, the N. caninum MIC1 protein has the amino acid sequence of SEQ ID NO:9. In another preferred embodiment, the N. caninum MAG1 protein has the amino acid sequence of SEQ ID NO:13. The present invention further provides substantially purified or isolated polypeptides that are homologous to any of the aforementioned N. caninum proteins. The present invention further provides polypeptides which are fusion proteins comprising any of the aforementioned polypeptides fused to a carrier or fusion partner, as known in the art. The present invention further provides polypeptides consisting of a substantial portion of any of the aforementioned polypeptides. The polypeptides of the present invention are useful both in vaccine compositions to protect mammals against neosporosis and as diagnostic reagents.
The present invention further provides a method of preparing any of the aforementioned polypeptides, comprising culturing host cells transformed with a recombinant expression vector, said vector comprising a polynucleotide molecule comprising a nucleotide sequence encoding any of the aforementioned polypeptides, wherein the nucleotide sequence is in operative association with one or more regulatory elements, under conditions conducive to the expression of the polypeptide, and recovering the expressed polypeptide from the cell culture.
The present invention further provides antibodies specifically directed against a N.caninum GRA1, GRA2, SAG1, MIC1 or MAG1 protein.
The present invention further provides genetic constructs for use in mutating a Neospora GRA1, GRA2, SAG1, MIC1 or MAG1 gene to produce modified Neospora cells. Such modified Neospora cells are useful in vaccine compositions to protect mammals against neosporosis. In a preferred though non-limiting embodiment, a genetic construct of the present invention comprises a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as a nucleotide sequence encoding a GRA1, GRA2, SAG1, MIC1 or MAG1 protein from N. caninum, or a substantial portion thereof, but that further comprises one or more mutations, i.e., one or more nucleotide deletions, insertions and/or substitutions, that can serve to mutate the gene. Once transformed into cells of Neospora, the polynucleotide molecule of the genetic construct is specifically targeted, e.g., by homologous recombination, to the particular Neospora gene, and either deletes or replaces the gene or a portion thereof, or inserts into the gene. As a result of this recombination event, the Neospora gene is mutated. The resulting mutated gene is preferably partially or fully disabled in that it encodes either a partially defective or fully defective protein, or fails to encode a protein. The present invention further provides Neospora cells which have been modified by one or more of said gene mutations, and methods of preparing modified Neospora cells using a genetic construct of the present invention.
The present invention further provides a vaccine against neosporosis, comprising an immunologically effective amount of a polypeptide of the present invention, or an immunologically effective amount of a polynucleotide molecule of the present invention, or an immunologically effective amount of modified Neospora cells of the present invention; and a veterinarily acceptable carrier. In a preferred embodiment, the vaccine of the present invention comprises modified live cells of N. caninum that express a GRA1xe2x88x92, GRA2xe2x88x92, SAG1xe2x88x92, MIC1xe2x88x92 or MAG1xe2x88x92 phenotype, or a combination of said phenotypes. In a non-limiting embodiment, the vaccine is a combination vaccine for protecting a mammal against neosporosis and, optionally, one or more other diseases or pathological conditions that can afflict the mammal, which combination vaccine comprises an immunologically effective amount of a first component comprising a polypeptide, polynucleotide molecule, or modified Neospora cells of the present invention; an immunologically effective amount of a second component that is different from the first component, and that is capable of inducing, or contributing to the induction of, a protective response against a disease or pathological condition that can afflict the mammal; and a veterinarily acceptable carrier.
The present invention further provides a method of preparing a vaccine against neosporosis, comprising combining an immunologically effective amount of a N. caninum polypeptide of the present invention, or an immunologically effective amount of a polynucleotide molecule of the present invention, or an immunologically effective amount of modified Neospora cells of the present invention, with a veterinarily acceptable carrier, in a form suitable for administration to a mammal.
The present invention further provides a method of vaccinating a mammal against neosporosis, comprising administering to the mammal an immunologically effective amount of a vaccine of the present invention.
The present invention further provides a kit for vaccinating a mammal against neosporosis, comprising a first container having an immunologically effective amount of a polypeptide of the present invention, or an immunologically effective amount of a polynucleotide molecule of the present invention, or an immunologically effective amount of modified Neospora cells of the present invention; and a second container having a veterinarily acceptable carrier or diluent.
An isolated polynucleotide molecule of the present invention can have a nucleotide sequence derived from any species or strain of Neospora, but is preferably from a pathogenic species of Neospora such as N. caninum. A non-limiting example of a strain of N. caninum from which a polynucleotide molecule of the present invention can be isolated or derived is strain NC-1, which is available in host MARC-145 monkey kidney cells under Accession No. CRL-12231 from the American Type Culture Collection (ATCC), located at 10801 University Blvd, Manassas, Va., 20110, USA. Strain NC-1 is also described in Dubey et al., 1988, J. Am. Vet. Med. Assoc. 193:1259-63, which publication is incorporated herein by reference. Alternatively, pathogenic strains or species of Neospora for use in practicing the present invention can be isolated from organs, tissues or body fluids of infected animals using standard isolation techniques such as those described in the publications reviewed above.
As used herein, the terms xe2x80x9cpolynucleotide molecule,xe2x80x9d xe2x80x9cpolynucleotide sequence,xe2x80x9d xe2x80x9ccoding sequence,xe2x80x9d xe2x80x9copen-reading frame (ORF),xe2x80x9d and the like, are intended to refer to both DNA and RNA molecules, which can either be single-stranded or double-stranded, and that can include one or more prokaryotic sequences, cDNA sequences, genomic DNA sequences including exons and introns, and chemically synthesized DNA and RNA sequences, and both sense and corresponding anti-sense strands. As used herein, the term xe2x80x9cORFxe2x80x9d refers to the minimal nucleotide sequence required to encode a particular Neospora protein, i.e., either a GRA1, GRA2, SAG1, MIC1 or MAG1 protein, without any intervening termination codons.
Production and manipulation of the polynucleotide molecules and oligonucleotide molecules disclosed herein are within the skill in the art and can be carried out according to recombinant techniques described, among other places, in Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al., 1989, Current Protocols In Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.; Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Innis et al. (eds), 1995, PCR Strategies, Academic Press, Inc., San Diego; and Erlich (ed), 1992, PCR Technology, Oxford University Press, New York, all of which are incorporated herein by reference.
References herein below to the nucleotide sequences shown in SEQ ID NOS:1 and 3, and to substantial portions thereof, are intended to also refer to the corresponding nucleotide sequences and substantial portions thereof, respectively, as present in plasmid pRC77 (ATCC 209685), unless otherwise indicated. In addition, references herein below to the amino acid sequences shown in SEQ ID NO:2, and to substantial portions and peptide fragments thereof, are intended to also refer to the corresponding amino acid sequences, and substantial portions and peptide fragments thereof, respectively, encoded by the corresponding GRA1-encoding nucleotide sequence present in plasmid pRC77 (ATCC 209685), unless otherwise indicated.
The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the GRA1 protein from N. caninum. In a preferred embodiment, the GRA1 protein has the amino acid sequence of SEQ ID NO:2. In a further preferred embodiment, the isolated GRA1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO:1 from about nt 205 to about nt 777, the nucleotide sequence of the open reading frame (ORF) of the GRA1 gene, which is presented in SEQ ID NO:3 from about nt 605 to about nt 1304, and the nucleotide sequence of the GRA1-encoding ORF of plasmid pRC77 (ATCC 209685). In a non-limiting embodiment, the isolated GRA1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:3. The GRA1 gene presented in SEQ ID NO:3 comprises an ORF from nt 605 to nt 855 and from nt 983 to nt 1304 with an intervening intron extending from nt 856 to nt 982. In addition, putative promoter motifs have been identified within 150 bp 5xe2x80x2 of the mRNA start site that are similar to those found in T. gondii GRA genes (see Section 5.3, below).
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a GRA1-encoding polynucleotide molecule of the present invention. The term xe2x80x9chomologousxe2x80x9d when used to refer to a GRA1-related polynucleotide molecule means a polynucleotide molecule having a nucleotide sequence: (a) that encodes the same protein as one of the aforementioned GRA1-encoding polynucleotide molecules of the present invention, but that includes one or more silent changes to the nucleotide sequence according to the degeneracy of the genetic code; or (b) that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the N. caninum GRA1 protein, under moderately stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65xc2x0 C., and washing in 0.2xc3x97SSC/0.1% SDS at 42xc2x0 C. (see Ausubel et al. (eds.), 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley and Sons, Inc., New York, at p. 2.10.3), and that is useful in practicing the present invention. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the N. caninum GRA1 protein under highly stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS 1 mM EDTA at 65xc2x0 C., and washing in 0.1xc3x97SSC/0. 1% SDS at 68xc2x0 C. (Ausubel et al., 1989, above), and is useful in practicing the present invention. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of a nucleotide sequence selected from the group consisting of the ORF of SEQ ID NO:1, which is from about nt 205 to about nt 777, and the ORF of the GRA1 gene, which is presented in SEQ ID NO:3 from about nt 605 to about nt 1304, and which is useful in practicing the present invention.
As used herein, a polynucleotide molecule is xe2x80x9cuseful in practicing the present inventionxe2x80x9d where the polynucleotide molecule can be used to amplify a Neospora-specific polynucleotide molecule using standard amplification techniques, or as a diagnostic reagent to detect the presence of a Neospora-specific polynucleotide in a fluid or tissue sample from a Neospora- infected animal.
Polynucleotide molecules of the present invention having nucleotide sequences that are homologous to the nucleotide sequence of a GRA1-encoding polynucleotide molecule of the present invention do not include polynucleotide molecules having the native nucleotide sequence of T. gondii encoding a T. gondii GRA protein, and further have no more than about 90%, and preferably no more than about 80%, sequence identity to such a T. gondii polynucleotide molecule, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, National Center for Biotechnology Information).
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the N. caninum GRA1 protein. As used herein to refer to polypeptides that are homologous to the N. caninum GRA1 protein, the term xe2x80x9chomologousxe2x80x9d refers to a polypeptide otherwise having the amino acid sequence of the N. caninum GRA1 protein, but in which one or more amino acid residues have been conservatively substituted with a different amino acid residue, where the resulting polypeptide is useful in practicing the present invention. Conservative amino acid substitutions are well-known in the art. Rules for making such substitutions include those described by Dayhof, M. D., 1978, Nat. Biomed. Res. Found., Washington, D.C., Vol. 5, Sup. 3, among others. More specifically, conservative amino acid substitutions are those that generally take place within a family of amino acids that are related in acidity, polarity, or bulkiness of their side chains. Genetically encoded amino acids are generally divided into four groups: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are also jointly classified as aromatic amino acids. One or more replacements within any particular group, e.g., of a leucine with an isoleucine or valine, or of an aspartate with a glutamate, or of a threonine with a serine, or of any other amino acid residue with a structurally related amino acid residue, e.g., an amino acid residue with similar acidity, polarity, bulkiness of side chain, or with similarity in some combination thereof, will generally have an insignificant effect on the function or immunogenicity of the polypeptide. In a preferred embodiment, the homologous polypeptide has at least about 70%, more preferably at least about 80%, and most preferably at least about 90% sequence identity to SEQ ID NO:2.
As used herein, a polypeptide is xe2x80x9cuseful in practicing the present inventionxe2x80x9d where the polypeptide can be used as a diagnostic reagent to detect the presence of Neospora-specific antibodies in a blood or serum sample from an animal that is currently infected, or that has been infected, with Neospora.
The present invention further provides a polynucleotide molecule consisting of a substantial portion of any of the aforementioned Neospora GRA1-related polynucleotide molecules of the present invention. As used herein, a xe2x80x9csubstantial portionxe2x80x9d of a GRA1-related polynucleotide molecule means a polynucleotide molecule consisting of less than the complete nucleotide sequence of the GRA1-related polynucleotide molecule, but comprising at least about 5%, more preferably at least about 10%, and most preferably at least about 20%, of the nucleotide sequence of the GRA1-related polynucleotide molecule, and that is useful in practicing the present invention, as usefulness is defined above for polynucleotide molecules.
In addition to the nucleotide sequences of any of the aforementioned GRA1-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences selected from those that naturally flank the GRA1 ORF or gene in situ in N. caninum, and include the nucleotide sequences shown in SEQ ID NO:1 from about nt 1 to about nt 204 and from about nt 778 to about nt 1265, or as shown in SEQ ID NO:3 from about nt 1 to about nt 604, and from about nt 1305 to about nt 1774, or substantial portions thereof.
References herein below to the nucleotide sequence shown in SEQ ID NO:4, and to substantial portions thereof, are intended to also refer to the corresponding nucleotide sequence and substantial portions thereof, respectively, as present in plasmid pRC5 (ATCC 209686), unless otherwise indicated. In addition, references herein below to the amino acid sequence shown in SEQ ID NO:5, and to substantial portions and peptide fragments thereof, are intended to also refer to the corresponding amino acid sequence, and substantial portions and peptide fragments thereof, respectively, encoded by the corresponding GRA2-encoding nucleotide sequence present in plasmid pRC5 (ATCC 209686), unless otherwise indicated.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the GRA2 protein from N. caninum. In a preferred embodiment, the GRA2 protein has the amino acid sequence of SEQ ID NO:5. In a further preferred embodiment, the isolated GRA2-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of the ORF of SEQ ID NO:4, which is from about nt 25 to about nt 660, and the nucleotide sequence of the GRA2-encoding ORF of plasmid pRC5 (ATCC 209686). In a non- limiting embodiment, the isolated GRA2-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:4.
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a GRA2-encoding polynucleotide molecule of the present invention. The term xe2x80x9chomologousxe2x80x9d when used to refer to a GRA2-related polynucleotide molecule means a polynucleotide molecule having a nucleotide sequence: (a) that encodes the same protein as one of the aforementioned GRA2-encoding polynucleotide molecules of the present invention, but that includes one or more silent changes to the nucleotide sequence according to the degeneracy of the genetic code; or (b) that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the N. caninum GRA2 protein, under moderately stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65xc2x0 C., and washing in 0.2xc3x97SSC/0.1% SDS at 42xc2x0 C. (Ausubel et aL, 1989, above), and that is useful in practicing the present invention, as usefulness is defined above for polynucleotide molecules. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the N. caninum GRA2 protein under highly stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65xc2x0 C., and washing in 0.1xc3x97SSC/0.1% SDS at68xc2x0 C. (Ausubel etal., 1989, above), and is useful in practicing the present invention. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of the nucleotide sequence of the ORF of SEQ ID NO:4, which is from about nt 25 to about nt 660, and is useful in practicing the present invention.
Polynucleotide molecules of the present invention having nucleotide sequences that are homologous to the nucleotide sequence of a GRA2-encoding polynucleotide molecule of the present invention do not include polynucleotide molecules having the native nucleotide sequence of T. gondii encoding a T. gondii GRA protein, and further have no more than about 90%, and preferably no more than about 80%, sequence identity to such a T gondii polynucleotide molecule, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, NCBI).
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the N. caninum GRA2 protein. As used herein to refer to polypeptides that are homologous to the N. caninum GRA2 protein, the term xe2x80x9chomologousxe2x80x9d refers to a polypeptide otherwise having the amino acid sequence of the N. caninum GRA2 protein, but in which one or more amino acid residues have been conservatively substituted with a different amino acid residue, as defined above, where the resulting polypeptide is useful in practicing the present invention, as.usefulness is defined above for polypeptides. In a preferred embodiment, the homologous polypeptide has at least about 70%, more preferably at least about 80%, and most preferably at least about 90% sequence identity to SEQ ID NO:5.
The present invention further provides a polynucleotide molecule consisting of a substantial portion of any of the aforementioned Neospora GRA2-related polynucleotide molecules of the present invention. As used herein, a xe2x80x9csubstantial portionxe2x80x9d of a GRA2-related polynucleotide molecule means a polynucleotide molecule consisting of less than the complete nucleotide sequence of the GRA2-related polynucleotide molecule, but comprising at least about 5%, more preferably at least about 10%, and most preferably at least about 20%, of the nucleotide sequence of the GRA2-related polynucleotide molecule, and that is useful in practicing the present invention, as usefulness is defined above for polynucleotide molecules.
In addition to the nucleotide sequences of any of the aforementioned GRA2-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences that naturally flank the GRA2 gene or ORF in situ in N. caninum, and include the flanking nucleotide sequences shown in SEQ ID NO:4 from about nt 1 to about nt 24, and from about nt 661 to about nt 1031, or substantial portions thereof.
References herein below to the nucleotide sequence shown in SEQ ID NO:6, and to substantial portions thereof, are intended to also refer to the corresponding nucleotide sequence and substantial portions thereof, respectively, as present in plasmid pRC102 (ATCC 209687), unless otherwise indicated. In addition, references herein below to the amino acid sequence shown in SEQ ID NO:7, and to substantial portions and peptide fragments thereof, are intended to also refer to the corresponding amino acid sequence, and substantial portions and peptide fragments thereof, respectively, encoded by the corresponding SAG1-encoding nucleotide sequence present in plasmid pRC102 (ATCC 209687), unless otherwise indicated.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the SAG1 protein from N. caninum. In a preferred embodiment, the SAG1 protein has the amino acid sequence of SEQ ID NO:7. In a further preferred embodiment, the isolated SAG1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of the ORF of SEQ ID NO:6, which is from about nt 130 to about nt 1089, and the nucleotide sequence of the SAG1-encoding ORF of plasmid pRC102 (ATCC 209687). In a non-limiting embodiment, the isolated SAG1-encoding polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:6.
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a SAG1-encoding polynucleotide molecule of the present invention. The term xe2x80x9chomologousxe2x80x9d when used to refer to a SAG1-related polynucleotide molecule means a polynucleotide molecule having a nucleotide sequence: (a) that encodes the same protein as one of the aforementioned SAG1-encoding polynucleotide molecules of the present invention, but that includes one or more silent changes to the nucleotide sequence according to the degeneracy of the genetic code; or (b) that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the N. caninum SAG1 protein, under moderately stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65xc2x0 C., and washing in 0.2xc3x97SSC/0.1% SDS at 42xc2x0 C. (Ausubel et al., 1989, above), and that is useful in practicing the present invention, as usefulness is defined above for polynucleotide molecules. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the N. caninum SAG1 protein under highly stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65xc2x0 C., and washing in 0.1xc3x97SSC/0.1% SDS at68xc2x0 C. (Ausubel etal., 1989, above), and is useful in practicing the present invention. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of the nucleotide sequence of the ORF of SEQ ID NO:6, which is from about nt 130 to about nt 1089, and is useful in practicing the present invention.
Polynucleotide molecules of the present invention having nucleotide sequences that are homologous to the nucleotide sequence of a SAG1-encoding polynucleotide molecule of the present invention do not include polynucleotide molecules having the native nucleotide sequence of T. gondii encoding a T. gondii SAG1 protein, and further have no more than about 90%, and preferably no more than about 80%, sequence identity to such a T. gondii polynucleotide molecule, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, NCBI).
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the N. caninum SAG1 protein. As used herein to refer to polypeptides that are homologous to the N. caninum SAG1 protein, the term xe2x80x9chomologousxe2x80x9d refers to a polypeptide otherwise having the amino acid sequence of the N. caninum SAG1 protein, but in which one or more amino acid residues have been conservatively substituted with a different amino acid residue, as defined above, where the resulting polypeptide is useful in practicing the present invention, as usefulness is defined above for polypeptides. In a preferred embodiment, the homologous polypeptide has at least about 70%, more preferably at least about 80%, and most preferably at least about 90% sequence identity to SEQ ID NO:7.
The present invention further provides a polynucleotide molecule consisting of a substantial portion of any of the aforementioned Neospora SAG1-related polynucleotide molecules of the present invention. As used herein, a xe2x80x9csubstantial portionxe2x80x9d of a SAG1-related polynucleotide molecule means a polynucleotide molecule consisting of less than the complete nucleotide sequence of the SAG1-related polynucleotide molecule, but comprising at least about 5%, more preferably at least about 10%, and most preferably at least about 20%, of the nucleotide sequence of the SAG1-related polynucleotide molecule, and that is useful in practicing the present invention, as usefulness is defined above for polynucleotide molecules.
In addition to the nucleotide sequences of any of the aforementioned SAG1-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences that naturally flank the SAG1 gene or ORF in situ in N. caninum, and include the flanking nucleotide sequences shown in SEQ ID NO:6 from about nt 1 to about nt 129 and from about nt 1090 to about nt 1263, or substantial portions thereof.
References herein below to the nucleotide sequences shown in SEQ ID NOS:8 and 10, and to substantial portions thereof, are intended to also refer to the corresponding nucleotide sequences and substantial portions thereof, respectively, as present in plasmid pRC340 (ATCC 209688), unless otherwise indicated. In addition, references herein below to the amino acid sequences shown in SEQ ID NO:9, and to substantial portions and peptide fragments thereof, are intended to also refer to the corresponding amino acid sequences, and substantial portions and peptide fragments thereof, respectively, encoded by the corresponding MIC1-encoding nucleotide sequence present in plasmid pRC340 (ATCC 209688), unless otherwise indicated.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the MIC1 protein from N. caninum. In a preferred embodiment, the MIC1 protein has the amino acid sequence of SEQ ID NO:9. In a further preferred embodiment, the isolated MIC1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of the ORF of SEQ ID NO:8, which is from about nt 138 to about nt 1520, the nucleotide sequence of the ORF of the MIC1 gene, which is presented as SEQ ID NO:10, and the nucleotide sequence of the MIC1-encoding ORF of plasmid pRC340 (ATCC 209688). In a non-limiting embodiment, the isolated MIC1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO:8 and SEQ ID NO:10.
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a MIC1-encoding polynucleotide molecule of the present invention. The term xe2x80x9chomologousxe2x80x9d when used to refer to a MIC1-related polynucleotide molecule means a polynucleotide molecule having a nucleotide sequence: (a) that encodes the same protein as one of the aforementioned MIC1-encoding polynucleotide molecules of the present invention, but that includes one or more silent changes to the nucleotide sequence according to the degeneracy of the genetic code; or (b) that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the N. caninum MIC1 protein, under moderately stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65xc2x0 C., and washing in 0.2xc3x97SSC/0.1% SDS at 42xc2x0 C. (Ausubel et al., 1989, above), and that is useful in practicing the present invention, as usefulness is defined above for polynucleotide molecules. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the N. caninum MIC1 protein under highly stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65xc2x0 C., and washing in 0.1 xc3x97SSC/0.1 % SDS at 68xc2x0 C. (Ausubel et al., 1989, above), and is useful in practicing the present invention. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of a nucleotide sequence selected from the group consisting of the ORF of SEQ ID NO:8 from about nt 138 to about nt 1520, and the ORF of the MIC1 gene, which is presented as SEQ ID NO:10, and is useful in practicing the present invention.
Polynucleotide molecules of the present invention having nucleotide sequences that are homologous to the nucleotide sequence of a MIC1-encoding polynucleotide molecule of the present invention do not include polynucleotide molecules having the native nucleotide sequence of T. gondii encoding a T. gondii MIC1 protein, and further have no more than about 90%, and preferably no more than about 80%, sequence identity to such a T. gondii polynucleotide molecule, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, NCBI).
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the N. caninum MIC1 protein. As used herein to refer to polypeptides that are homologous to the N. caninum MIC1 protein, the term xe2x80x9chomologousxe2x80x9d refers to a polypeptide otherwise having the amino acid sequence of the N. caninum MIC1 protein, but in which one or more amino acid residues have been conservatively substituted with a different amino acid residue, as defined above, where the resulting polypeptide is useful in practicing the present invention, as usefulness is defined above for polypeptides. In a preferred embodiment, the homologous polypeptide has at least about 70%, more preferably at least about 80%, and most preferably at least about 90% sequence identity to SEQ ID NO:9.
The present invention further provides a polynucleotide molecule consisting of a substantial portion of any of the aforementioned Neospora MIC1-related polynucleotide molecules of the present invention. As used herein, a xe2x80x9csubstantial portionxe2x80x9d of a MIC1-related polynucleotide molecule means a polynucleotide molecule consisting of less than the complete nucleotide sequence of the MIC1-related polynucleotide molecule, but comprising at least about 5%, more preferably at least about 10%, and most preferably at least about 20%, of the nucleotide sequence of the MIC1-related polynucleotide molecule, and that is useful in practicing the present invention, as usefulness is defined above for polynucleotide molecules.
In addition to the nucleotide sequences of any of the aforementioned MIC1-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences that naturally flank the MIC1 ORF or gene in situ in N. caninum, and include the nucleotide sequences as shown in SEQ ID NO:8 from about nt 1 to about 137, and from about nt 1521 to about nt 2069, or substantial portions thereof.
References herein below to the nucleotide sequence shown in SEQ ID NO:11, and to substantial portions thereof, are intended to also refer to the corresponding nucleotide sequences and substantial portions thereof, respectively, as present in plasmid bd304 (ATCC 203413), unless otherwise indicated. In addition, references herein below to the amino acid sequence shown in SEQ ID NO:13, and to substantial portions and peptide fragments thereof, are intended to also refer to the corresponding amino acid sequence, and substantial portions and peptide fragments thereof, respectively, encoded by the corresponding MAG1-encoding nucleotide sequence present in plasmid bd304 (ATCC 203413), unless otherwise indicated.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the MAG1 protein from N. caninum. In a preferred embodiment, the MAG1 protein has the amino acid sequence of SEQ ID NO:13. In a further preferred embodiment, the isolated MAG1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence presented in SEQ ID NO:11 from about nt 1305 to about nt 2786, a cDNA molecule prepared therefrom, such as a cDNA molecule having the ORF of SEQ ID NO:12 from about nt 122 to about nt 1381, and the nucleotide sequence of the MAG1-encoding ORF present in plasmid bd304 (ATCC 203413). The present invention further provides a polynucleotide molecule having a nucleotide sequence of any ORF present in SEQ ID NO:11. In a non-limiting embodiment, the isolated MAG1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO:11 and a cDNA deduced therefrom based on the putative exon/intron boundaries.
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a MAG1-encoding polynucleotide molecule of the present invention. The term xe2x80x9chomologousxe2x80x9d when used to refer to a MAG1-related polynucleotide molecule means a polynucleotide molecule having a nucleotide sequence: (a) that encodes the same protein as one of the aforementioned MAG1-encoding polynucleotide molecules of the present invention, but that includes one or more silent changes to the nucleotide sequence according to the degeneracy of the genetic code; or (b) that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the N. caninum MAG1 protein, under moderately stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65xc2x0 C., and washing in 0.2xc3x97SSC/0.1% SDS at 42xc2x0 C. (Ausubel et al., 1989, above), and that is useful in practicing the present invention, as usefulness is defined above for polynucleotide molecules. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the N. caninum MAG1 protein under highly stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65xc2x0 C., and washing in 0.1xc3x97SSC/0.1 % SDS at 68xc2x0 C. (Ausubel et al., 1989, above), and is useful in practicing the present invention. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of a nucleotide sequence selected from the group consisting of the nucleotide sequence of the ORF of the MAG1 gene, which is presented in SEQ ID NO:11 from about nt 1305 to about nt 2786 and a cDNA molecule prepared therefrom based on the putative exon/intron boundaries, such as a cDNA molecule having the ORF of SEQ ID NO:12 from about nt 122 to about nt 1381, and is useful in practicing the present invention.
Polynucleotide molecules of the present invention having nucleotide sequences that are homologous to the nucleotide sequence of a MAG1-encoding polynucleotide molecule of the present invention do not include polynucleotide molecules having the native nucleotide sequence of T. gondii encoding a T. gondii MAG1 protein, and further have no more than about 90%, and preferably no more than about 80%, sequence identity to such a T. gondii polynucleotide molecule, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, NCBI).
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the N. caninum MAG1 protein. As used herein to refer to polypeptides that are homologous to the N. caninum MAG1 protein, the term xe2x80x9chomologousxe2x80x9d refers to a polypeptide otherwise having the amino acid sequence of the N. caninum MAG1 protein, but in which one or more amino acid residues have been conservatively substituted with a different amino acid residue, as defined above, where the resulting polypeptide is useful in practicing the present invention, as usefulness is defined above for polypeptides. In a preferred embodiment, the homologous polypeptide has at least about 70%, more preferably at least about 80%, and most preferably at least about 90% sequence identity to SEQ ID NO:13.
The present invention further provides a polynucleotide molecule consisting of a substantial portion of any of the aforementioned Neospora MAG1-related polynucleotide molecules of the present invention. As used herein, a xe2x80x9csubstantial portionxe2x80x9d of a MAG1-related polynucleotide molecule means a polynucleotide molecule consisting of less than the complete nucleotide sequence of the MAG1-related polynucleotide molecule, but comprising at least about 5%, more preferably at least about 10%, and most preferably at least about 20%, of the nucleotide sequence of the MAG1-related polynucleotide molecule, and that is useful in practicing the present invention, as usefulness is defined above for polynucleotide molecules. For example, a substantial portion of the polynucleotide molecule of SEQ ID NO:11 can comprise putative exon 1 from about nt 704 to about nt 820, or putative exon 2 from about nt 1301 to about nt 1399, or putative exon 3 from about nt 1510 to about nt 1808, or putative exon 4 from about nt 1921 to about nt 3297.
In addition to the nucleotide sequences of any of the aforementioned MAG1-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences that naturally flank the MAG1 gene or ORF in situ in N. caninum, and include the nucleotide sequences as shown in SEQ ID NO:11 from about nt 1 to about nt 1304, and from about nt 2787 to about nt 4242, or that naturally flank the ORF of a cDNA molecule prepared therefrom based on the putative exon/intron boundaries, and include flanking sequences of the ORF of a cDNA molecule having the ORF of SEQ ID NO:12, from about nt 1 to about nt 121, and from about nt 1382 to about nt 1892, or substantial portions thereof.
The present invention further provides a polynucleotide molecule comprising the nucleotide sequence of the N. caninum GRA1 and MAG1 gene promoters. During the conduct of the experimental work disclosed herein, it was determined that the N. caninum GRA1 and MAG1 genes disclosed herein are naturally arranged in situ in a head-to-head orientation with an intervening nucleotide sequence of about 577 nt in length. This intervening nucleotide sequence, which is presented in SEQ ID NO:11 from nt 127 to nt 703, represents a putative bidirectional promoter region comprising the promoters of both the N. caninum GRA1 and MAG1 genes.
The GRA1IMAG1 bidirectional promoter region of the present invention, and functional portions thereof, are useful for a variety of purposes including for controlling the recombinant expression of either the GRA1 or MAG1 genes, or both genes, or of one or more other genes or coding sequences, in host cells of N. caninum or in host cells of any other species of Neospora or other member of the Apicomplexa, or in any other appropriate host cell. Such other genes or coding sequences can either be native or heterologous to the recombinant host cell. The promoter sequence can be fused to the particular gene or coding sequence using standard recombinant techniques as known in the art so that the promoter sequence is in operative association therewith, as xe2x80x9coperative associationxe2x80x9d is defined below. By using the promoter, recombinant expression systems can be constructed and used to screen for compounds and transcriptional factors that can modulate the expression of the GRA1 and MAG1 genes of Neospora or other members of the Apicomplexa. In addition, such promoter constructs can be used to express heterologous polypeptides in Neospora or other members of the Apicomplexa.
The present invention further provides oligonucleotide molecules that hybridize to any one of the aforementioned polynucleotide molecules of the present invention, or that hybridize to a polynucleotide molecule having a nucleotide sequence that is the complement of any one of the aforementioned polynucleotide molecules of the present invention. Such oligonucleotide molecules are preferably at least about 10 nucleotides in length, and more preferably from about 15 to about 30 nucleotides in length, and hybridize to one or more of the aforementioned polynucleotide molecules under highly stringent conditions, i.e., washing in 6xc3x97SSC/0.5% sodium pyrophosphate at about 37xc2x0 C. for xcx9c14-base oligos, at about 48xc2x0 C. for xcx9c17-base oligos, at about 55xc2x0 C. for xcx9c20-base oligos, and at about 60xc2x0 C. for xcx9c23-base oligos. Other hybridization conditions for longer oligonucleotide molecules of the present invention can be determined by the skilled artisan using standard techniques. In a preferred embodiment, an oligonucleotide molecule of the present invention is complementary to a portion of at least one of the aforementioned polynucleotide molecules of the present invention.
Specific though non-limiting embodiments of oligonucleotide molecules useful in practicing the present invention include oligonucleotide molecules selected from the group consisting of SEQ ID NOS:14-26 and 28-34, and the complements thereof.
The oligonucleotide molecules of the present invention are useful for a variety of purposes, including as primers in amplification of a Neospora-specific polynucleotide molecule for use, e.g., in differential disease diagnosis, or to encode or act as antisense molecules useful in gene regulation. Regarding diagnostics, suitably designed primers can be used to detect the presence of Neospora-specific polynucleotide molecules in a sample of animal tissue or fluid, such as brain tissue, lung tissue, placental tissue, blood, cerebrospinal fluid, mucous, urine, amniotic fluid, etc. The production of a specific amplification product can support a diagnosis of Neospora infection, while lack of an amplified product can point to a lack of infection. Methods for conducting amplifications, such as the polymerase chain reaction (PCR), are described, among other places, in Innis et al. (eds), 1995, above; and Erlich (ed), 1992, above. Other amplification techniques known in the art, e.g., the ligase chain reaction, can alternatively be used. The sequences of the polynucleotide molecules disclosed herein can also be used to design primers for use in isolating homologous genes from other species or strains of Neospora or other members of the Apicomplexa.
The present invention further provides compositions for cloning and expressing any of the polynucleotide molecules of.the present invention, including cloning vectors, expression vectors, transformed host cells comprising any of said vectors, and novel strains or cell lines derived therefrom. In a preferred embodiment, the present invention provides a recombinant vector comprising a polynucleotide molecule having a nucleotide sequence encoding the GRA1, GRA2, SAG1, MIC1 or MAG1 protein of N. caninum. In specific though non-limiting embodiments, the present invention provides plasmid pRC77 (ATCC 209685), which encodes the N. caninum GRA1 protein; plasmid pRC5 (ATCC 209686), which encodes the N. caninum GRA2 protein; plasmid pRC102 (ATCC 209687), which encodes the N. caninum SAG1 protein; plasmid pRC340 (ATCC 209688), which encodes the N. caninum MIC1 protein; and plasmid bd304 (ATCC 203413), which encodes the N. caninum MAG1 protein, and which also comprises the bidirectional promoter region described above.
Recombinant vectors of the present invention, particularly expression vectors, are preferably constructed so that the coding sequence for the polynucleotide molecule of the invention is in operative association with one or more regulatory elements necessary for transcription and translation of the coding sequence to produce a polypeptide. As used herein, the term xe2x80x9cregulatory elementxe2x80x9d includes but is not limited to nucleotide sequences that encode inducible and non-inducible promoters, enhancers, operators and other elements known in the art that serve to drive and/or regulate expression of polynucleotide coding sequences. Also, as used herein, the coding sequence is in xe2x80x9coperative associationxe2x80x9d with one or more regulatory elements where the regulatory elements effectively regulate and allow for the transcription of the coding sequence or the translation of its mRNA, or both.
Methods are well-known in the art for constructing recombinant vectors containing particular coding sequences in operative association with appropriate regulatory elements, and these can be used to practice the present invention. These methods include in vitro recombinant techniques, synthetic techniques, and in vivo genetic recombination. See, e.g., the techniques described in Maniatis et al., 1989, above; Ausubel et al., 1989, above; Sambrook et al., 1989, above; Innis et al., 1995, above; and Erlich, 1992, above.
A variety of expression vectors are known in the art which can be utilized to express the GRA1, GRA2, SAG1, MIC1, and MAG1 coding sequences of the present invention, including recombinant bacteriophage DNA, plasmid DNA, and cosmid DNA expression vectors containing the particular coding sequences. Typical prokaryotic expression vector plasmids that can be engineered to contain a polynucleotide molecule of the present invention include pUC8, pUC9, pBR322 and pBR329 (Biorad Laboratories, Richmond, Calif.), pPL and pKK223 (Pharmacia, Piscataway, N.J.), pQE50 (Qiagen, Chatsworth, Calif.), and pGEM-T EASY (Promega, Madison, Wis.), among many others. Typical eukaryotic expression vectors that can be engineered to contain a polynucleotide molecule of the present invention include an ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, Calif.), cytomegalovirus promoter-enhancer-based systems (Promega, Madison, Wis.; Stratagene, La Jolla, Calif.; Invitrogen), and baculovirus-based expression systems (Promega), among others.
The regulatory elements of these and other vectors can vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements can be used. For instance, when cloning in mammalian cell systems, promoters isolated from the genome of mammalian cells, e.g., mouse metallothionein promoter, or from viruses that grow in these cells, e.g., vaccinia virus 7.5 K promoter or Moloney murine sarcoma virus long terminal repeat, can be used. Promoters obtained by recombinant DNA or synthetic techniques can also be used to provide for transcription of the inserted sequence. In addition, expression from certain promoters can be elevated in the presence of particular inducers, e.g., zinc and cadmium ions for metallothionein promoters. Non-limiting examples of transcriptional regulatory regions or promoters include for bacteria, the xcex2-gal promoter, the T7 promoter, the TAC promoter, xcex left and right promoters, trp and lac promoters, trp-lac fusion promoters, etc.; for yeast, glycolytic enzyme promoters, such as ADH-II and -II promoters, GPK promoter, PGI promoter, TRP promoter, etc.; and for mammalian cells, SV40 early and late promoters, adenovirus major late promoters, among others. The present invention further provides a polynucleotide molecule comprising the nucleotide sequence of the promoters of both the GRA1 and MAG1 genes of N. caninum, which can be used to express any of the coding sequences of the present invention in Neospora or other members of the Apicomplexa.
Specific initiation signals are also required for sufficient translation of inserted coding sequences. These signals typically include an ATG initiation codon and adjacent sequences. In cases where the polynucleotide molecule of the present invention including its own initiation codon and adjacent sequences are inserted into the appropriate expression vector, no additional translation control signals may be needed. However, in cases where only a portion of a coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, may be required. These exogenous translational control signals and initiation codons can be obtained from a variety of sources, both natural and synthetic. Furthermore, the initiation codon must be in phase with the reading frame of the coding regions to ensure in- frame translation of the entire insert.
Expression vectors can also be constructed that will express a fusion protein comprising a protein or polypeptide of the present invention. Such fusion proteins can be used, e.g., to raise antisera against a Neospora protein, to study the biochemical properties of the Neospora protein, to engineer a Neospora protein exhibiting different immunological or functional properties, or to aid in the identification or purification, or to improve the stability, of a recombinantly-expressed Neospora protein. Possible fusion protein expression vectors include but are not limited to vectors incorporating sequences that encode xcex2-galactosidase and trpE fusions, maltose-binding protein fusions, glutathione-S-transferase fusions and polyhistidine fusions (carrier regions). Methods are well-known in the art that can be used to construct expression vectors encoding these and other fusion proteins.
The fusion protein can be useful to aid in purification of the expressed protein. In non-limiting embodiments, e.g., a GRA1-maltose-binding fusion protein can be purified using amylose resin; a GRA1-glutathione-S-transferase fusion protein can be purified using glutathione-agarose beads; and a GRA1-polyhistidine fusion protein can be purified using divalent nickel resin. Alternatively, antibodies against a carrier protein or peptide can be used for affinity chromatography purification of the fusion protein. For example, a nucleotide sequence coding for the target epitope of a monoclonal antibody can be engineered into the expression vector in operative association with the regulatory elements and situated so that the expressed epitope is fused to a Neospora protein of the present invention. In a non-limiting embodiment, a nucleotide sequence coding for the FLAG(trademark) epitope tag (International Biotechnologies Inc.), which is a hydrophilic marker peptide, can be inserted by standard techniques into the expression vector at a point corresponding, e.g., to the amino or carboxyl terminus of the GRA1 protein. The expressed GRA1 protein-FLAG(trademark) epitope fusion product can then be detected and affinity-purified using commercially available anti-FLAG(trademark) antibodies.
The expression vector can also be engineered to contain polylinker sequences that encode specific protease cleavage sites so that the expressed Neospora protein can be released from the carrier region or fusion partner by treatment with a specific protease. For example, the fusion protein vector can include a nucleotide sequence encoding a thrombin or factor Xa cleavage site, among others.
A signal sequence upstream from and in reading frame with the Neospora coding sequence can be engineered into the expression vector by known methods to direct the trafficking and secretion of the expressed protein. Non-limiting examples of signal sequences include those from xcex1-factor, immunoglobulins, outer membrane proteins, penicillinase, and T-cell receptors, among others.
To aid in the selection of host cells transformed or transfected with a recombinant vector of the present invention, the vector can be engineered to further comprise a coding sequence for a reporter gene product or other selectable marker. Such a coding sequence is preferably in operative association with the regulatory elements, as described above. Reporter genes that are useful in practicing the invention are well-known in the art and include those encoding chloramphenicol acetyltransferase (CAT), green fluorescent protein, firefly luciferase, and human growth hormone, among others. Nucleotide sequences encoding selectable markers are well-known in the art, and include those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of such sequences include those that encode thymidine kinase activity, or resistance to methotrexate, ampicillin, kanamycin, chloramphenicol, zeocin, pyrimethamine, aminoglycosides, or hygromycin, among others.
The present invention further provides transformed host cells comprising a polynucleotide molecule or recombinant vector of the present invention, and cell lines derived therefrom. Host cells useful in practicing the invention can be eukaryotic or prokaryotic cells. Such transformed host cells include but are not limited to microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA vectors, or yeast transformed with a recombinant vector, or animal cells, such as insect cells infected with a recombinant virus vector, e.g., baculovirus, or mammalian cells infected with a recombinant virus vector, e.g., adenovirus or vaccinia virus, among others. For example, a strain of E. coli can be used, such as, e.g., the DH5xcex1 strain available from the ATCC, Rockville, Md., USA (Accession No. 31343), or from Stratagene (La Jolla, Calif.). Eukaryotic host cells include yeast cells, although mammalian cells, e.g., from a mouse, hamster, cow, monkey, or human cell line, among others, can also be utilized effectively. Examples of eukaryotic host cells that can be used to express a recombinant protein of the invention include Chinese hamster ovary (CHO) cells (e.g., ATCC Accession No. CCL-61), NIH Swiss mouse embryo cells NIH/3T3 (e.g., ATCC Accession No. CRL-1658), and Madin-Darby bovine kidney (MDBK) cells (ATCC Accession No. CCL-22).
The recombinant vector of the invention is preferably transformed or transfected into one or more host cells of a substantially homogeneous culture of cells. The vector is generally introduced into host cells in accordance with known techniques, such as, e.g., by protoplast transformation, calcium phosphate precipitation, calcium chloride treatment, microinjection, electroporation, transfection by contact with a recombined virus, liposome-mediated transfection, DEAE-dextran transfection, transduction, conjugation, or microprojectile bombardment, among others. Selection of transformants can be conducted by standard procedures, such as by selecting for cells expressing a selectable marker, e.g., antibiotic resistance, associated with the recombinant expression vector.
Once an expression vector is introduced into the host cell, the integration and maintenance of the polynucleotide molecule of the present invention, either in the host cell genome or episomally, can be confirmed by standard techniques, e.g., by Southern hybridization analysis, restriction enzyme analysis, PCR analysis including reverse transcriptase PCR (rt-PCR), or by immunological assay to detect the expected protein product. Host cells containing and/or expressing a polynucleotide molecule of the present invention can be identified by any of at least four general approaches that are well-known in the art, including: (i) DNA-DNA, DNA-RNA, or RNA-antisense RNA hybridization; (ii) detecting the presence of xe2x80x9cmarkerxe2x80x9d gene functions; (iii) assessing the level of transcription as measured by the expression of specific mRNA transcripts in the host cell; or (iv) detecting the presence of mature polypeptide product, e.g., by immunoassay, as known in the art.
Once a polynucleotide molecule of the present invention has been stably introduced into an appropriate host cell, the transformed host cell is clonally propagated, and the resulting cells are grown under conditions conducive to the maximum production of the encoded polypeptide. Such conditions typically include growing transformed cells to high density. Where the expression vector comprises an inducible promoter, appropriate induction conditions such as, e.g., temperature shift, exhaustion of nutrients, addition of gratuitous inducers (e.g., analogs of carbohydrates, such as isopropyl-xcex2-D-thiogalactopyranoside (IPTG)), accumulation of excess metabolic by-products, or the like, are employed as needed to induce expression.
Where the polypeptide is retained inside the host cells, the cells are harvested and lysed, and the product is substantially purified or isolated from the lysate under extraction conditions known in the art to minimize protein degradation such as, e.g., at 4xc2x0 C., or in the presence of protease inhibitors, or both. Where the polypeptide is secreted from the host cells, the exhausted nutrient medium can simply be collected and the polypeptide substantially purified or isolated therefrom.
The polypeptide can be substantially purified or isolated from cell lysates or culture medium, as necessary, using standard methods, including but not limited to one or more of the following methods: ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density centrifugation, and affinity chromatography. If the polypeptide lacks biological activity, it can. be detected as based, e.g., on size, or reactivity with a polypeptide-specific antibody, or by the presence of a fusion tag. For use in practicing the present invention, the polypeptide can be in an unpurified state as secreted into the culture fluid or as present in a cell lysate, but is preferably substantially purified or isolated therefrom. As used herein, a polypeptide is xe2x80x9csubstantially purifiedxe2x80x9d where the polypeptide constitutes at least about 20 wt % of the protein in a particular preparation. Also, as used herein, a polypeptide is xe2x80x9cisolatedxe2x80x9d where the polypeptide constitutes at least about 80 wt% of the protein in a particular preparation.
Thus, the present invention provides a substantially purified or isolated polypeptide encoded by a polynucleotide of the present invention. In a non-limiting embodiment, the polypeptide is a N. caninum protein selected from the group consisting of GRA1, GRA2, SAG1, MIC1 and MAG1 proteins. In a preferred embodiment, the N. caninum GRA1 protein has the amino acid sequence of SEQ ID NO:2. In another preferred embodiment, the N. caninum GRA2 protein has the amino acid sequence of SEQ ID NO:5. In another preferred embodiment, the N. caninum SAG1 protein has the amino acid sequence of SEQ ID NO:7. In another preferred embodiment, the N. caninum MIC1 protein has the amino acid sequence of SEQ ID NO:9. In another preferred embodiment, the N. caninum MAG1 protein has the amino acid sequence of SEQ ID NO:13.
The present invention further provides polypeptides that are homologous to any of the aforementioned N. caninum proteins, as the term xe2x80x9chomologousxe2x80x9d is defined above for polypeptides. Polypeptides of the present invention that are homologous to any of the aforementioned GRA1, GRA2, SAG1, MIC1 or MAG1 proteins of N. caninum do not include polypeptides having the native amino acid sequence of a T. gondii GRA, SAG, MIC or MAG protein, and further have no more than about 90%, and preferably no more than about 80%, amino acid sequence identity to such a T. gondii polypeptide, wherein sequence identity is determined by use of the BLASTP algorithm (GenBank, NCBI).
The present invention further provides polypeptides consisting of a substantial portion of any one of the aforementioned polypeptides of the present invention. As used herein, a xe2x80x9csubstantial portionxe2x80x9d of a polypeptide of the present invention, or xe2x80x9cpeptide fragment,xe2x80x9d means a polypeptide consisting of less than the complete amino acid sequence of the corresponding full-length polypeptide, but comprising at least about 10%, and more preferably at least about 20%, of the amino acid sequence thereof, and that is useful in practicing the present invention, as defined above for polypeptides. Particularly preferred are peptide fragments that are immunogenic, i.e., capable of inducing an immune response which results in production of antibodies that react specifically against the corresponding full-length Neospora polypeptide.
The present invention further provides fusion proteins comprising any of the aforementioned polypeptides fused to a carrier or fusion partner as known in the art.
The present invention further provides a method of preparing any of the aforementioned polypeptides, comprising culturing a host cell transformed with a recombinant expression vector, said recombinant expression vector comprising a polynucleotide molecule comprising a nucleotide sequence encoding the particular polypeptide, which polynucleotide molecule is in operative association with one or more regulatory elements, under conditions conducive to the expression of the polypeptide, and recovering the expressed polypeptide from the cell culture.
Once a polypeptide of the present invention of sufficient purity has been obtained, it can be characterized by standard methods, including by SDS-PAGE, size exclusion chromatography, amino acid sequence analysis, immunological activity, biological activity, etc. The polypeptide can be further characterized using hydrophilicity analysis (see, e.g., Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824), or analogous software algorithms, to identify hydrophobic and hydrophilic regions. Structural analysis can be carried out to identify regions of the polypeptide that assume specific secondary structures. Biophysical methods such as X-ray crystallography (Engstrom, 1974, Biochem. Exp. Biol. 11: 7-13), computer modeling (Fletterick and Zoller (eds), 1986, in: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and nuclear magnetic resonance (NMR) can be used to map and study potential sites of interaction between the polypeptide and other putative interacting proteins/receptors/molecules. Information obtained from these studies can be used to design deletion mutants and vaccine compositions, and to design or select therapeutic or pharmacologic compounds that can specifically block the biological function of the polypeptide in vivo.
Polypeptides of the present invention are useful for a variety of purposes, including as components of vaccine compositions to protect mammals against neosporosis; or as diagnostic reagents, e.g., using standard techniques such as ELISA assays, to screen for Neospora-specific antibodies in blood or serum samples from animals; or as antigens to raise polyclonal or monoclonal antibodies, as described below, which antibodies are useful as diagnostic reagents, e.g., using standard techniques such as Western blot assays, to screen for Neospora-specific proteins in cell, tissue or fluid samples from an animal.
Any polypeptide of the present invention can be modified at the protein level to improve or otherwise alter its biological or immunological characteristics. One or more chemical modifications of the polypeptide can be carried out using known techniques to prepare analogs therefrom, including but not limited to any of the following: substitution of one or more L-amino acids of the polypeptide with corresponding D-amino acids, amino acid analogs, or amino acid mimics, so as to produce, e.g., carbazates or tertiary centers; or specific chemical modification, such as, e.g., proteolytic cleavage with trypsin, chymotrypsin, papain or V8 protease, or treatment with NaBH4 or cyanogen bromide, or acetylation, formylation, oxidation or reduction, etc. Alternatively or additionally, polypeptides of the present invention can be modified by genetic recombination techniques.
A polypeptide of the present invention can be derivatized by conjugation thereto of one or more chemical groups, including but not limited to acetyl groups, sulfur bridging groups, glycosyl groups, lipids, and phosphates, and/or by conjugation to a second polypeptide of the present invention, or to another protein, such as, e.g., serum albumin, keyhole limpet hemocyanin, or commercially activated BSA, or to a polyamino acid (e.g., polylysine), or to a polysaccharide, (e.g., sepharose, agarose, or modified or unmodified celluloses), among others. Such conjugation is preferably by covalent linkage at amino acid side chains and/or at the N-terminus or C-terminus of the polypeptide. Methods for carrying out such conjugation reactions are well-known in the field of protein chemistry.
Derivatives useful in practicing the claimed invention also include those in which a water-soluble polymer such as, e.g., polyethylene glycol, is conjugated to a polypeptide of the present invention, or to an analog or derivative thereof, thereby providing additional desirable properties while retaining, at least in part, the immunogenicity of the polypeptide. These additional desirable properties include, e.g., increased solubility in aqueous solutions, increased stability in storage, increased resistance to proteolytic degradation, and increased in vivo half-life. Water-soluble polymers suitable for conjugation to a polypeptide of the present invention include but are not limited to polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ethers, and xcex1,xcex2-poly[2-hydroxyethyl]-DL-aspartamide. Polyethylene glycol is particularly preferred. Methods for making water-soluble polymer conjugates of polypeptides are known in the art and are described in, among other places, U.S. Pat. No. 3,788,948; U.S. Pat. No. 3,960,830; U.S. Pat. No. 4,002,531; U.S. Pat. No. 4,055,635; U.S. Pat. No. 4,179,337; U.S. Pat. No. 4,261,973; U.S. Pat. No. 4,412,989; U.S. Pat. No. 4,414,147; U.S. Pat. No. 4,415,665; U.S. Pat. No. 4,609,546; U.S. Pat. No. 4,732,863; U.S. Pat. No. 4,745,180; European Patent (EP) 152,847; EP 98,110; and Japanese Patent 5,792,435, which patents are incorporated herein by reference.
The present invention further provides isolated antibodies directed against a polypeptide of the present invention. In a preferred embodiment, antibodies can be raised against a GRA1, GRA2, SAG1, MIC1 or MAG1 protein from N. caninum using known methods. Various host animals selected from pigs, cows, horses, rabbits, goats, sheep, or mice, can be immunized with a partially or substantially purified, or isolated, N. caninum protein, or with a homolog, fusion protein, substantial portion, analog or derivative thereof, as these are described above. An adjuvant, such as described below, can be used to enhance antibody production.
Polyclonal antibodies can be obtained and isolated from the serum of an immunized animal and tested for specificity against the antigen using standard techniques. Alternatively, monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (Nature, 1975, 256: 495-497); the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030); and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, techniques described for the production of single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted to produce N. caninum antigen-specific single chain antibodies. These publications are incorporated herein by reference.
Antibody fragments that contain specific binding sites for a polypeptide of the present invention are also encompassed within the present invention, and can be generated by known techniques. Such fragments include but are not limited to F(abxe2x80x2)2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(abxe2x80x2)2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., 1989, Science 246: 1275-1281) to allow rapid identification of Fab fragments having the desired specificity to the N. caninum protein.
Techniques for the production and isolation of monoclonal antibodies and antibody fragments are well-known in the art, and are additionally described, among other places, in Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, and in J. W. Goding, 1986, Monoclonal Antibodies: Principles and Practice, Academic Press, London, which are incorporated herein by reference.
Based on the disclosure of the polynucleotide molecules of the present invention, genetic constructs can be prepared for use in disabling or otherwise mutating a Neospora GRA1, GRA2, SAG1, MIC1 or MAG1 gene (which genes are hereinafter referred to collectively or individually as the xe2x80x9cNeospora genesxe2x80x9d or a xe2x80x9cNeospora gene,xe2x80x9d respectively). Each of the Neospora genes can be mutated using an appropriately designed genetic construct in combination with genetic techniques now known or to be developed in the future. For example, a Neospora gene can be mutated using a genetic construct of the present invention that functions to: (a) delete all or a portion of the coding sequence or regulatory sequence of the Neospora gene; or (b) replace all or a portion of the coding sequence or regulatory sequence of the Neospora gene with a different nucleotide sequence; or (c) insert into the coding sequence or regulatory sequence of the Neospora gene one or more nucleotides, or an oligonucleotide molecule, or polynucleotide molecule, which can comprise a nucleotide sequence from Neospora or from a heterologous source; or (d) carry out some combination of (a), (b) and (c).
Neospora cells in which a Neospora gene has been mutated are useful in practicing the present invention where mutating the gene reduces the pathogenicity of the Neospora cells carrying the mutated gene compared to cells of the same strain of Neospora where the gene has not been so mutated, and where such Neospora cells carrying the disabled gene can be used in a vaccine composition, particularly in a modified live vaccine, to induce or contribute to the induction of, a protective response in a mammal against neosporosis. In a preferred embodiment, the mutation serves to partially or completely disable the Neospora gene, or partially or completely disable the protein encoded by the Neospora gene. In this context, a Neospora gene or protein is considered to be partially or completely disabled if either no protein product is made (for example, the gene is deleted), or a protein product is made that can no longer carry out its normal biological function or can no longer be transported to its normal cellular location, or a product is made that carries out its normal biological function but at a significantly reduced rate, or if such mutation results in a detectable decrease in the pathogenicity of cells of a pathogenic strain of Neospora wherein the gene has been so mutated compared to cells of the same strain but in which the gene has not be so mutated.
In a non-limiting embodiment, a genetic construct of the present invention is used to mutate a wild-type Neospora gene by replacement of the coding sequence of the wild-type gene, or a promoter or other regulatory region thereof, or a portion thereof, with a different nucleotide sequence such as, e.g., a mutated coding sequence or mutated regulatory region, or portion thereof. Mutated Neospora gene sequences for use in such a genetic construct can be produced by any of a variety of known methods, including by use of error-prone PCR, or by cassette mutagenesis. For example, oligonucleotide-directed mutagenesis can be employed to alter the coding sequence or promoter sequence of a wild-type Neospora gene in a defined way, e.g., to introduce a frame-shift or a termination codon at a specific point within the sequence. Alternatively or additionally, a mutated nucleotide sequence for use in the genetic construct of the present invention can be prepared by insertion into the coding sequence or promoter sequence of one or more nucleotides, oligonucleotide molecules or polynucleotide molecules, or by replacement of a portion of the coding sequence or promoter sequence with one or more different nucleotides, oligonucleotide molecules or polynucleotide molecules. Such oligonucleotide molecules or polynucleotide molecules can be obtained from any naturally occurring source or can be synthetic. The inserted sequence can serve simply to disrupt the reading frame of the Neospora gene, or can further encode a heterologous gene product such as a selectable marker.
Alternatively or additionally, random mutagenesis can be used to produce a mutated Neospora gene sequence for use in a genetic construct of the present invention. Random mutagenesis can be carried out by any techniques now known or to be developed in the future such as, e.g., by exposing cells carrying a Neospora gene to ultraviolet radiation or x-rays, or to chemical mutagens such as N-methyl-Nxe2x80x2-nitrosoguanidine, ethyl methane sulfonate, nitrous acid or nitrogen mustards, and then selecting for cells carrying a mutation in the particular gene. See, e.g., Ausubel, 1989, above, for a review of mutagenesis techniques.
Mutations to produce modified Neospora cells that are useful in practicing the present invention, as defined above, can occur anywhere in the Neospora gene, including in the ORF, or in the promoter or other regulatory region, or in any other sequences that naturally comprise the gene or ORF. Such Neospora cells include mutants in which a modified form of the protein normally encoded by the Neospora gene is produced, or in which no protein normally encoded by the Neospora gene is produced, and can be null, conditional or leaky mutants.
Alternatively, a genetic construct of the present invention can comprise nucleotide sequences that naturally flank the Neospora gene or ORF in situ, such as those presented in SEQ ID NOS:1, 3, 4, 6, 8, 10, 11 and 12, with only a portion or no nucleotide sequences from the coding region of the gene itself. Such a genetic construct would be useful, e.g., to delete the entire Neospora gene or ORF.
In a preferred embodiment, a genetic construct of the present invention comprises a polynucleotide molecule that can be used to disable a Neospora gene, comprising: (a) a polynucleotide molecule having a nucleotide sequence that is otherwise the same as a nucleotide sequence encoding a GRA1, GRA2, SAG1, MIC1 or MAG1 protein from N. caninum, but which nucleotide sequence further comprises one or more disabling mutations; or (b) a polynucleotide molecule comprising a nucleotide sequence that naturally flanks the ORF of a Neospora gene in situ. Once transformed into cells of a strain of Neospora, the polynucleotide molecule of the genetic construct is specifically targeted to the particular Neospora gene by homologous recombination, and thereby either replaces the gene or portion thereof or inserts into the gene. As a result of this recombination event, the Neospora gene otherwise native to that particular strain of Neospora is disabled.
Methods for carrying out homologous gene replacement in parasitic protozoans are known in the art, and are described, among other places, in Cruz and Beverley, 1990, Nature 348:171-173; Cruz et al., 1991, Proc. Natl. Acad. Sci. USA 88:7170-7174; Donald and Roos, 1994, Mol. Biochem. Parasitol. 63:243-253; and Titus et al., 1995, Proc. Natl. Acad. Sci. USA 92:10267-10271, all of which are incorporated herein by reference.
For targeted gene mutation through homologous recombination, the genetic construct is preferably a plasmid, either circular or linearized, comprising a mutated nucleotide sequence as described above. In a non-limiting embodiment, at least about 200 nucleotides of the mutated sequence are used to specifically direct the genetic construct of the present invention to the particular targeted Neospora gene for homologous recombination, although shorter lengths of nucleotides can also be effective. In addition, the plasmid preferably comprises an additional nucleotide sequence encoding a reporter gene product or other selectable marker that is constructed so that it will insert into the Neospora genome in operative association with the regulatory element sequences of the native Neospora gene to be disrupted. Reporter genes that can be used in practicing the invention are well-known in the art and include those encoding CAT, green fluorescent protein, and xcex2-galactosidase, among others. Nucleotide sequences encoding selectable markers are also well-known in the art, and include those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of such sequences include those that encode pyrimethamine resistance, or neomycin phosphotransferase (which confers resistance to aminoglycosides), or hygromycin phosphotransferase (which confers resistance to hygromycin).
Methods that can be used for creating the genetic constructs of the present invention are well-known in the art, and include in vitro recombinant techniques, synthetic techniques, and in vivo genetic recombination, as described, among other places, in Maniatis et al., 1989, above; Ausubel et al., 1989, above; Sambrook et al., 1989, above; Innis et al., 1995, above; and Erlich, 1992, above.
Neospora cells can be transformed or transfected with a genetic construct of the present invention in accordance with known techniques, such as, e.g., by electroporation. Selection of transformants can be carried out using standard techniques, such as by selecting for cells expressing a selectable marker associated with the construct. Identification of transformants in which a successful recombination event has occurred and the particular target gene has been disabled can be carried out by genetic analysis, such as by Southern blot analysis, or by Northern analysis to detect a lack of mRNA transcripts encoding the particular protein, or by the appearance of a novel phenotype, such as reduced pathogenicity, or cells lacking the particular protein, as determined, e.g., by immunological analysis, or some combination thereof.
Neospora cells that can be modified according to the present invention are preferably tachyzoites, but can alternatively be bradyzoites or oocysts. Although cells in certain stages of the Neospora life cycle are diploid, tachyzoites are haploid. Thus, the use of tachyzoites in the production of modified Neospora cells expressing the appropriate mutant phenotype is preferred because tachyzoites require only a single successful recombination event to disrupt the particular Neospora gene. Alternatively, in diploid cells of Neospora, two alleles must be disrupted for each gene. This can be accomplished by sequentially targeting the first allele and then the second allele with genetic constructs bearing two different selectable markers.
In a further non-limiting embodiment, the genetic construct of the present invention can additionally comprise a different gene or coding region from Neospora or from a different pathogen that infects the animal, which gene or coding region encodes an antigen useful to induce, or contribute to the induction of, a separate and distinct protective immune response in the animal upon vaccination with the modified live Neospora cells of the present invention. This additional gene or coding region can be further engineered to contain a signal sequence that leads to secretion of the encoded antigen from the modified live Neospora cell, thereby allowing for the antigen to be displayed to the immune system of the vaccinated animal.
The present invention thus provides modified live Neospora cells in which the GRA1, GRA2, SAG1, MIC1 or MAG1 gene has been mutated. The present invention further provides modified live Neospora cells in which a combination of two or more of the GRA1, GRA2, SAG1, MIC1, and MAG1 genes have been mutated, which cells can be prepared using the general methods presented above. In addition, the present invention provides a method of preparing modified live Neospora cells, comprising: (a) transforming cells of Neospora with a genetic construct of the invention; (b) selecting transformed cells in which the GRA1, GRA2, SAG1, MIC1, or MAG1 gene has been mutated by the genetic construct; and (c) selecting from among the cells of step (b) those cells that can be used in a vaccine to protect a mammal against neosporosis.
Neospora cells for use in the present invention can be cultured and maintained in vitro by infecting any receptive host cell line, preferably a mammalian cell line, with tachyzoites according to known techniques described in the art. Mammalian cell lines in which tachyzoites of Neospora can be cultured include, e.g., human foreskin fibroblasts (Lindsay et al., 1993, Am. J. Vet. Res. 54:103-106), bovine cardiopulmonary aortic endothelial cells (Marsh et al., 1995, above), bovine monocytes (Lindsay and Dubey, 1989, above), and monkey kidney cells, among others. For example, tachyzoites of N. caninum can be cultured in monolayers of Hs68 human foreskin fibroblast cells (ATCC Accession No. CRL-1635) (Lindsay et al, 1993, above); and MARC145 monkey kidney cells infected with tachyzoites of N. caninum strain NC-1 for use in the present invention are on deposit with the ATCC (Accession No. 12231). Bradyzoites can be similarly cultured and manipulated.
Mammalian cell cultures can be grown, and cell cultures that have been infected with Neospora cells can be maintained, in any of several types of culture media described in the art. For example, stationary monolayer cultures of bovine cardiopulmonary aortic endothelial cells infected with tachyzoites of N. caninum can be grown in Dulbecco""s Minimum Essential Medium (DMEM; Gibco Laboratories, N.Y.), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) or adult equine serum (ES), 2 mM L-glutamine, 50 U/ml penicillin, and 50 xcexcg/ml streptomycin (Conrad et al., 1993, above). Monolayers of Hs68 human foreskin fibroblast cells can be maintained in RPMI 1640 containing 2% (v/v) FBS, 1.0 mM sodium pyruvate, 1xc3x97104 U/ml penicillin, 1xc3x97104 xcexcg/mI streptomycin, 5xc3x97102 mM 2-mercaptoethanol and 0.3 mg/ml L-glutamine (maintenance medium). Monolayer cultures of Hs68 human foreskin fibroblast cells infected with Neospora can be maintained in identical media, but in which the FBS is increased to 10% (v/v) (growth medium).
Neospora-infected monolayer cultures of mammalian cells are typically maintained under standard tissue culture conditions such as, e.g., at 37xc2x0 C. and 5% CO2. Tachyzoites are typically passaged to uninfected monolayer cultures when 70-90% of the mammalian cells in the culture have become infected, which can be determined microscopically using standard techniques. Tachyzoites can be collected from the infected mammalian cell cultures by lysing the host cells using any standard technique and collecting the tachyzoites, e.g., by filtration or by centrifugation.
Modified live Neospora cells of the present invention can also be cultured in mammalian cells, as described above.
The present invention further provides a vaccine against neosporosis, comprising an immunologically effective amount of one or more proteins or polypeptides of the present invention, and a veterinarily acceptable carrier. In a preferred embodiment, the vaccine comprises a N. caninum protein selected from the group consisting of GRA1, GRA2, SAG1, MIC1 and MAG1.
The present invention further provides a vaccine against neosporosis, comprising an immunologically effective amount of one or more polynucleotide molecules of the present invention, and a veterinarily acceptable carrier. In a preferred embodiment, the vaccine comprises a polynucleotide molecule having a nucleotide sequence encoding a N. caninum protein selected from the group consisting of GRA1, GRA2, SAG1, MIC1, and MAG1.
The present invention further provides a vaccine against neosporosis, comprising an immunologically effective amount of modified Neospora cells of the present invention, and a veterinarily acceptable carrier. In a preferred embodiment, the modified Neospora cells for use in the vaccine of the present invention are live cells of N. caninum which express a GRA1, GRA2xe2x88x92, SAG1xe2x88x92, MIC1xe2x88x92, or MAG1xe2x88x92 phenotype. Alternatively, the vaccine of the present invention can comprise any of such modified Neospora cells of the present invention that have been inactivated. Inactivation of modified Neospora cells can be carried out using any techniques known in the art, including by chemical treatment, such as with binary ethylenimine (BEI), or beta-propiolactone, or by freeze-thawing or heat treatment, or by homogenization of cells, or by a combination of these types of techniques. Vaccines prepared from homogenized, modified Neospora cells can consist of either the entire unfractionated cell homogenate, or an immunologically effective subfraction thereof. As used herein, the term xe2x80x9cimmunologically effective amountxe2x80x9d refers to that amount of antigen, e.g., protein, polypeptide, polynucleotide molecule, or modified cells, capable of inducing a protective response against neosporosis when administered to a member of a mammalian species after either a single administration, or after multiple administrations.
The phrase xe2x80x9ccapable of inducing a protective responsexe2x80x9d is used broadly herein to include the induction or enhancement of any immune-based response in the animal in response to vaccination, including either an antibody or cell-mediated immune response, or both, that serves to protect the vaccinated animal against neosporosis. The terms xe2x80x9cprotective responsexe2x80x9d and xe2x80x9cprotectxe2x80x9d as used herein refer not only to the absolute prevention of neosporosis or absolute prevention of infection by a neosporosis-causing pathogen, but also to any detectable reduction in the degree or rate of infection by such a pathogen, or any detectable reduction in the severity of the disease or any symptom or condition resulting from infection by the pathogen, including, e.g., any detectable reduction in the rate of formation, or in the absolute number, of lesions formed in one or more tissues, or any detectable reduction in the occurrence of abortion, or the transmission of infection from a pregnant mammal to its fetus or from a mammal parent to its offspring, in the vaccinated animal as compared to an unvaccinated infected animal of the same species.
In a further preferred embodiment, the vaccine of the present invention is a combination vaccine for protecting a mammal against neosporosis and, optionally, one or more other diseases or pathological conditions that can afflict the mammal, which combination vaccine comprises an immunologically effective amount of a first component comprising a polypeptide, polynucleotide molecule, or modified Neospora cells of the present invention; an immunologically effective amount of a second component that is different from the first component, and that is capable of inducing, or contributing to the induction of, a protective response against a disease or pathological condition that can afflict the mammal; and a veterinarily acceptable carrier.
The second component of the combination vaccine is selected based on its ability to induce, or contribute to the induction of, a protective response against either neosporosis or another disease or pathological condition that can afflict members of the mammalian species, as known in the art. Any antigenic component now known in the art, or to be determined in the future, to be useful in a vaccine composition in the particular mammalian species can be used as the second component of the combination vaccine. Such antigenic components include but are not limited to those that provide protection against pathogens selected from the group consisting of bovine herpes virus (syn., infectious bovine rhinotracheitis), bovine respiratory syncitial virus, bovine viral diarrhea virus, parainfluenza virus types I, II, or III, Leptospira spp., Campylobacter spp., Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma spp., Klebsiella spp., Salmonella spp., rotavirus, coronavirus, rabies, Pasteurella hemolytica, Pasteurella multocida, Clostridia spp., Tetanus toxoid, E. coli, Cryptosporidium spp., Eimeria spp., Trichomonas spp., and other eukaryotic parasites, among others.
In a non-limiting embodiment, the combination vaccine of the present invention comprises a combination of two or more components selected from the group consisting of an immunologically effective amount of a protein or polypeptide of the present invention, an immunologically effective amount of a polynucleotide molecule of the present invention, and an immunologically effective amount of modified Neospora cells of the present invention. In a preferred embodiment, the combination vaccine of the present invention comprises a combination of two or more components selected from the group consisting of N. caninum GRA1, GRA2, SAG1, MIC1, and MAG1 proteins, polynucleotide molecules encoding any of the N. caninum GRA1, GRA2, SAG1, MIC1, and MAG1 proteins, and modified live Neospora cells exhibiting any of the GRA1xe2x88x92, GRA2xe2x88x92, SAG1xe2x88x92, MIC1xe2x88x92, and MAG1xe2x88x92 phenotypes.
The vaccines of the present invention can further comprise one or more additional immunomodulatory components including, e.g., an adjuvant or cytokine, as described below.
The present invention further provides a method of preparing a vaccine against neosporosis, comprising combining an immunologically effective amount of a N. caninum protein or polypeptide, or polynucleotide molecule, or modified Neospora cells of the present invention, with a veterinarily acceptable carrier, in a form suitable for administration to a mammal. In a preferred embodiment, the protein is a N. caninum protein selected from the group consisting of GRA1, GRA2, SAG1, MIC1 and MAG1; the polynucleotide molecule preferably comprises a nucleotide sequence encoding a N. caninum protein selected from the group consisting of GRA1, GRA2, SAG1, MIC1 and MAG1; and the modified Neospora cells preferably are live cells that exhibit a phenotype selected from the group consisting of GRA1xe2x88x92, GRA2xe2x88x92, SAG1xe2x88x92, MIC1xe2x88x92, and MAG1xe2x88x92.
A vaccine comprising modified live Neospora cells of the present invention can be prepared using an aliquot of culture fluid containing said Neospora cells, either free in the medium or residing in mammalian host cells, or both, and can be administered directly or in concentrated form to the mammal. Alternatively, modified live Neospora cells can be combined with a veterinarily acceptable carrier, with or without an immunomodulatory agent, selected from those known in the art and appropriate to the chosen route of administration, preferably where at least some degree of viability of the modified live Neospora cells in the vaccine composition is maintained. Modified Neospora cells that can be used in the vaccine of the present invention are preferably tachyzoites, but can alternatively be bradyzoites or oocysts, or some combination thereof.
Vaccine compositions of the present invention can be formulated following accepted convention to include veterinarily acceptable carriers, such as standard buffers, stabilizers, diluents, preservatives, and/or solubilizers, and can also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Suitable other vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art,. See, e.g., Remington""s Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.
The vaccine of the present invention can further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cytokine, among others. Non-limiting examples of adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund""s complete and incomplete adjuvants, Block co polymer (CytRx, Atlanta GA), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.), AMPHIGEN(copyright) adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, and Avridine lipid-amine adjuvant. Specific non-limiting examples of oil-in-water emulsions useful in the vaccine of the invention include modified SEAM62 and SEAM xc2xd formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1% (v/v) SPAN(copyright) 85 detergent (ICI Surfactants), 0.7% (v/v) TWEEN(copyright) detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 xcexcg/ml Quil A, 100 xcexcg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM xc2xd is an oil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v) SPAN(copyright) 85 detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol, 100 xcexcg/ml Quil A, and 50 xcexcg/ml cholesterol. Other immunomodulatory agents that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines. Where the vaccine comprises modified live Neospora cells, the adjuvant is preferably selected based on the ability of the resulting vaccine formulation to maintain at least some degree of viability of the modified live Neospora cells.
Where the vaccine composition comprises a polynucleotide molecule, the polynucleotide molecule can either be DNA or RNA, although DNA is preferred, and is preferably administered to a mammal to be protected against neosporosis in an expression vector construct, such as a recombinant plasmid or viral vector, as known in the art. Examples of recombinant viral vectors include recombinant adenovirus vectors and recombinant retrovirus vectors. However, a preferred vaccine formulation comprises a non-viral DNA vector, most preferably a DNA plasmid-based vector. The polynucleotide molecule may be associated with lipids to form, e.g., DNA-lipid complexes, such as liposomes or cochleates. See, e.g., International Patent Publication WO 93/24640.
An expression vector useful as a vaccinal agent in a DNA vaccine preferably comprises a nucleotide sequence encoding one or more antigenic Neospora proteins, or a substantial portion of such a nucleotide sequence, in operative association with one or more transcriptional regulatory elements required for expression of the Neospora coding sequence in a eukaryotic cell, such as, e.g., a promoter sequence, as known in the art. In a preferred embodiment, the regulatory element is a strong viral promoter such as, e.g., a viral promoter from RSV or CMV. Such an expression vector also preferably includes a bacterial origin of replication and a prokaryotic selectable marker gene for cloning purposes, and a polyadenylation sequence to ensure appropriate termination of the expressed mRNA. A signal sequence may also be included to direct cellular secretion of the expressed protein.
The requirements for expression vectors useful as vaccinal agents in DNA vaccines are further described in U.S. Pat. No. 5,703,055, U.S. Pat. No. 5,580,859, U.S. Pat. No. 5,589,466, International Patent Publication WO 98/35562, and in various scientific publications, including Ramsay et al., 1997, Immunol. Cell Biol. 75:360-363; Davis, 1997, Cur. Opinion Biotech. 8:635-640; Maniackan et al., 1997, Critical Rev. Immunol. 17:139-154; Robinson, 1997, Vaccine 15(8):785-787; Lai and Bennett, 1998, Critical Rev. Immunol. 18:449-484; and Vogel and Sarver, 1995, Clin. Microbiol. Rev. 8(3):406-410, among others.
Where the vaccine composition comprises modified live Neospora cells, the vaccine can be stored cold or frozen. Where the vaccine composition instead comprises a protein, polypeptide, polynucleotide molecule, or inactivated modified Neospora cells of the present invention, the vaccine may be stored frozen, or in lyophilized form to be rehydrated prior to administration using an appropriate diluent.
The vaccine of the present invention can optionally be formulated for sustained release of the antigen. Examples of such sustained release formulations include antigen in combination with composites of biocompatible polymers, such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including A. Domb et al., 1992, Polymers for Advanced Technologies 3: 279-292, which is incorporated herein by reference. Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in the text by M. Chasin and R. Langer (eds), 1990, xe2x80x9cBiodegradable Polymers as Drug Delivery Systemsxe2x80x9d in: Drugs and the Pharmaceutical Sciences, Vol. 45, M. Dekker, N.Y., which is also incorporated herein by reference. Alternatively, or additionally, the antigen can be microencapsulated to improve administration and efficacy. Methods for microencapsulating antigens are well-known in the art, and include techniques described, e.g., in U.S. Pat. No. 3,137,631; U.S. Pat. No. 3,959,457; U.S. Pat. No. 4,205,060; U.S. Pat. NO. 4,606,940; U.S. Pat. No. 4,744,933; U.S. Pat. No. 5,132,117; and International Patent Publication WO 95/28227, all of which are incorporated herein by reference.
Liposomes can also be used to provide for the sustained release of antigen. Details concerning how to make and use liposomal formulations can be found in, among other places, U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050; and U.S. Pat. No. 5,009,956, all of which are incorporated herein by reference.
The present invention further provides a method of vaccinating a mammal against neosporosis, comprising administering to the mammal an immunologically effective amount of a vaccine of the present invention. The vaccine is preferably administered parenterally, e.g., either by subcutaneous or intramuscular injection. However, the vaccine can alternatively be administered by intraperitoneal or intravenous injection, or by other routes, including, e.g., orally, intranasally, rectally, vaginally, intra-ocularly, or by a combination of routes, and also by delayed release devices as known in the art. The skilled artisan will be able to determine the most optimal route of vaccine administration, and will also recognize acceptable formulations for the vaccine composition according to the chosen route of administration.
An effective dosage can be determined by conventional means, starting with a low dose of antigen, and then increasing the dosage while monitoring the effects. Numerous factors may be taken into consideration when determining an optimal dose per animal. Primary among these is the species, size, age and general condition of the animal, the presence of other drugs in the animal, the virulence of a particular species or strain of Neospora against which the animal is being vaccinated, and the like. The actual dosage is preferably chosen after consideration of the results from other animal studies.
The dose amount of a Neospora protein or polypeptide of the present invention in a vaccine of the present invention preferably ranges from about 10 xcexcg to about 10 mg, more preferably from about 50 xcexcg to about 1 mg, and most preferably from about 100 xcexcg to about 0.5 mg. The dose amount of a Neospora polynucleotide molecule of the present invention in a vaccine of the present invention preferably ranges from about 50 xcexcg to about 1 mg. The dose amount of modified Neospora cells of the present invention in a vaccine of the present invention preferably ranges from about 1xc3x97103 to about 1xc3x97101 cells/ml, and more preferably from about 1xc3x97105 to about 1xc3x97107 cells/ml. A suitable dosage size ranges from about 0.5 ml to about 10 ml, and more preferably from about 1 ml to about 5 ml. The dose amounts of these antigens are also applicable to combination vaccines of the present invention. Where the second component of the combination vaccine is an antigen other than a Neospora protein, polypeptide, polynucleotide or modified cell of the present invention, the dose amount of the second component for use in the combination vaccine can be determined from prior vaccine applications of that second component, as known in the art.
The vaccine of the present invention is useful to protect mammals against neosporosis. As used herein, the term xe2x80x9cmammalxe2x80x9d refers to any mammalian species that can be protected against neosporosis using the vaccine of the invention, including dogs, cows, goats, sheep and horses, among others. The vaccine of the invention can be administered at any time during the life of a particular animal depending upon several factors including, e.g., the timing of an outbreak of neosporosis among other animals, etc. The vaccine can be administered to animals of weaning age or younger, or to more mature animals, e.g., as a pre-breeding vaccine to protect against Neospora-related congenital disease or abortion. Effective protection may require only a primary vaccination, or one or more booster vaccinations may also be needed. One method of detecting whether adequate immune protection has been achieved is to determine seroconversion and antibody titer in the animal after vaccination. The timing of vaccination and the number of boosters, if any, will preferably be determined by a veterinarian based on analysis of all relevant factors, some of which are described above.
The present invention further provides a kit for vaccinating a mammal against neosporosis, comprising a container having an immunologically effective amount of a polypeptide, polynucleotide molecule, or modified Neospora cells of the present invention, or a combination thereof. The kit can optionally comprise a second container having a veterinarily acceptable carrier or diluent. In a preferred embodiment, the polypeptide is selected from the group consisting of GRA1, GRA2, SAG1, MIC1 and MAG1 proteins of N. caninum; the polynucleotide molecule preferably has a nucleotide sequence that encodes a N. caninum protein selected from the group consisting of GRA1, GRA2, SAG1, MIC1, and MAG1; and the modified Neospora cells preferably are live cells that express a GRA1xe2x88x92, GRA2xe2x88x92, SAG1xe2x88x92, MIC1xe2x88x92 or MAG1xe2x88x92 phenotype.
The following example is illustrative only, and is not intended to limit the scope of the present invention.